Project Summary Ehrlichia chaffeensis is a gram-negative, obligately intracellular bacterium that is the causative agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. A major knowledge gap is in our understanding of the mechanisms whereby E. chaffeensis establishes intracellular infection of the mononuclear phagocyte and avoids innate host defenses. We recently demonstrated that E. chaffeensis utilizes a type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors that interact with host cell DNA and a functionally diverse array of host proteins involved in transcriptional and translational regulation, post translational modification, signaling, immune response, intracellular trafficking, cytoskeletal organization and apoptosis. Two TRP120 interacting host proteins include the Notch activating metalloprotease ADAM17, and the negative regulator, FBW7. Notch is a highly conserved eukaryotic signaling pathway that plays a key role in determining cell growth, differentiation and function, which extends to immune cells. Recent studies have identified a functional relationship between the Notch pathway and negative regulation of toll-like receptor (TLR)-mediated immune responses and autophagy, both of which are suppressed during E. chaffeensis infection. Preliminary studies that serve as the basis for this proposal demonstrate that E. chaffeensis activates canonical Notch signaling pathway to promote survival. Activation of this pathway promotes ehrlichial survival by downregulating PU.1 (transcription factor required for TLR2/4 expression) and TLR expression, and by inhibiting of lysosomal fusion and autophagic degradation. The objective of this proposal is to determine the role of Ehrlichia TRPs in Notch pathway activation, and to demonstrate TRP-induced Notch-mediated modulation of PRR expression and autophagy. We hypothesize E. chaffeensis activates the Notch pathway through molecular interactions between TRP effectors and the Notch receptor complex to initiate a prosurvival cell signaling program that suppresses innate defenses. We will test this hypothesis with the following specific aims: (1) Determine the effect of E. chaffeensis induced Notch signaling on innate host defenses; (2) Define the mechanism of E. chaffeensis TRP-mediated Notch pathway activation and regulation. This investigation will expand our knowledge regarding exploitation of highly conserved host cell pathways by E. chaffeensis effectors in order to facilitate bacterial intracellular survival. Moreover, new targets for the development of novel therapeutic approaches against E. chaffeensis infection will be identified.